Conjugated linoleic acid-producing strains of probiotic bacteria and use thereof for the preparation of a food, dietetic or pharmaceutical composition

ABSTRACT

The present invention relates to conjugated linoleic acid (CLA)-producing strains of probiotic bacteria. In particular, the present invention relates to a selection of bacterial strains belonging to the genus Bifidobacterium which were selected for their ability to produce conjugated linoleic acid (CLA) from linoleic acid (LA). Furthermore, the present invention relates to a food, dietetic or pharmaceutical composition comprising said bacterial strains capable of increasing the quantity of CLA in situ, i.e. inside the gastrointestinal tract.

RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 13/516,579, filed on Dec. 17, 2012; which is a U.S.national stage application filed under 35 U.S.C. §371, based onInternational Application No. PCT/IB2010/003244, filed on Dec. 14, 2010,which claims the benefit of priority to Italian Patent Application Nos.RM2009A000662, filed on Dec. 16, 2009, and MI2010A002235, filed on Dec.3, 2010. The entire contents of the foregoing applications are herebyincorporated herein by reference.

The present invention relates to conjugated linoleic acid(CLA)-producing strains of probiotic bacteria. In particular, thepresent invention relates to a selection of bacterial strains belongingto the genus Bifidobacterium which were selected for their ability toproduce conjugated linoleic acid (CLA) from linoleic acid (LA).Furthermore, the present invention relates to a food, dietetic orpharmaceutical composition comprising said bacterial strains and capableof increasing the quantity of CLA in situ, i.e. inside thegastrointestinal tract.

The use of conjugated linoleic acid (CLA) in the preparation of foodproducts and supplements is known.

The development of said preparations is tied to the beneficial effectsprovided by CLA inside the body.

However, in the past thirty years, the dietary intake of CLA hasdrastically decreased for two fundamental reasons.

Firstly, the consumption of pork and milk derivatives, which representthe principal sources of these molecules, has greatly decreased.

Secondly, modern livestock rearing techniques have led pasture grass andforage to be replaced with industrially produced feedstuffs, which donot contain much natural linoleic acid.

Adding CLA to a food substrate to preparare CLA-rich/supplemented foodsis known. However, said food products enriched/supplemented with CLApresent a number of drawbacks and limits of application due to the factthat the oral administration thereof does not always represent a validand effective way of delivering CLA into the body; in particular the insitu delivery of CLA in the gastrointestinal tract of interest is verycompromised and limited. Moreover, in some countries the direct additionof CLA, as an active ingredient, to food products is not always allowed.

There is thus a need to have a composition which is capable ofovercoming the limits of the prior art and of representing a valid,effective means of delivering CLA into the body.

The excess of linoleic acid in the modern human diet is due not only toa low consumption of fish, but also to a high consumption of productsrich in vegetable oils and seeds. This situation is not, moreover,characteristic solely of the human diet, but rather also extends to thediet of animals used for food, which are commonly fed grain.

All this is reflected in a worse ratio between ω6 fatty acids and ω3fatty acids in meat products, dairy products and eggs, which are amongthe foods most widely consumed in the diet.

However, an improvement in the ω6/ω3 ratio by dietary means alone todaypresents a number of difficulties and problems unlikely to be overcome,since said problems are tied to the production and marketing systems,which are managed on an industrial scale and thus difficult to modify.

Therefore, it becomes necessary to be able to reduce the quantity oflinoleic acid (LA) deriving from the diet, which accumulates in excessin the body.

The subject matter of the present invention relates to a selection ofstrains of probiotic bacteria capable of converting linoleic acid (LA)into conjugated linoleic acid (CLA), as claimed in the appended claim.

The subject matter of the present invention further relates to a food ordietetic or pharmaceutical composition, as claimed in the appendedclaim.

The subject matter of the present invention further relates to apharmaceutical composition for use as a medication, as claimed in theappended claim.

The subject matter of the present invention further relates to the useof at least one bacterial strain capable of converting LA into CLA forthe preparation of a composition, as claimed in the appended claim.

Further preferred embodiments of the present invention will be describedand illustrated below, without being in any way intended to limit thescope of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the growth curves in time of the strain B. breve DSM 20213in TPY and MRS broth at different concentrations of LA (0, 0.5 and 1mg/ml). The initial pH for the TPY culture broth is 6.11; for MRS it is5.83. The final pH values are given in the figure itself.

FIG. 2, Table 1 shows the qualitative and quantitative composition ofthree culture media used in the present invention: TPY, MRS and LATPg.FIG. 2, Table 2 shows the absorbance values obtained from thespectrophotometric reading at a wavelength of 233 nm with respect to theconcentrations of CLA.

FIG. 3, Table 3 shows the regression line obtained with the values shownin table 2. FIG. 3, Table 4 shows the pour plate counts obtained usingthe TPY and MRS culture media at different concentrations of LA.

FIG. 4, Table 5 shows the values obtained for the concentration ofCLA/ml produced; percentage of LA-CLA conversion (expressed as CLAproduced/0.50 mg/ml LA); CLA concentration ratio/OD 600 nm. FIG. 4,Table 6 shows the quantification of CLA present in the TPY culturemedium with and without inoculation of LA.

FIG. 5, Table 7 shows the resistance of selected strains to gastricjuices, bile and pancreatic secretion.

All strains have been deposited in accordance with the Treaty ofBudapest and are accessible to the public on request from the competentDepositing Authority. Such Depositing Authorities include DSMZ (DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH; Inhoffenstr. 7B,D-38124 Braunschweig, Germany).

By virtue of intense research activity, the Applicant has succeeded inselecting specific strains of probiotic bacteria, belonging to the genusBifidobacterium.

Advantageously, said strains belong to the species B. longum and B.breve.

In particular, the strains selected by the Applicant are selected fromthe group comprising:

-   -   Bifidobacterium longum, deposited with the DSMZ and having the        accession number DSM 23233, filing date 12 Dec. 2010, depositor        Probiotical S.p.A.;    -   Bifidobacterium breve, deposited with the DSMZ and having the        accession number DSM 16604, filing date 20 Jul. 2004, depositor        Probiotical S.p.A.;    -   Bifidobacterium breve, deposited with the DSMZ and having the        accession number DSM 16596, filing date 21 Jul. 2004, depositor        Probiotical S.p.A.;    -   Bifidobacterium breve, deposited with the DSMZ and having the        accession number DSM 20213.

The Applicant has surprisingly found that the selected strains belongingto the genus Bifidobacterium are capable of converting linoleic acid(LA) into conjugated linoleic acid (CLA), with a conversion rate greaterthan 65%.

Advantageously, the selected strains are capable of converting linoleicacid (LA) into conjugated linoleic acid (CLA), with a conversion rategreater than 70%; even more advantageously, greater than 90%.

In the context of the present invention, LA means an omega-6 unsaturatedfatty acid called [cis, cis-9,12-octadecadienoic acid] CAS N. 60-33-3(also known as 18:2(n-6)).

In the context of the present invention, CLA means a group of at least28 isomers of linoleic acid found, for example, in meat and in dairyproducts. The group comprises the isomer [cis-9,trans-11] and the isomer[trans-10,cis-12], among others.

In the context of the present invention, conversion of LA into CLArefers to the obtainment of a mixture comprising the isomer[cis-9,trans-11] and the isomer [trans-10,cis-12], where the former ispresent in larger quantity or, alternatively, to the obtainment solelyof the isomer [cis-9,trans-11].

All of the strains tested by the Appliicant are of human origin and wereisolated from faecal material.

Moreover, the selected strains were also tested to verify theirresistance to gastric juices, bile and pancreatic secretion.

The results are shown in table 7.

In table 7, (*) indicates the survival of probiotic strains in twodifferent types of gastric juices and in a simulated pancreaticsecretion at 37° C. after different contact times (5 and 30 minutes).

In table 7, (**) indicates the results of survival in the presence ofbiliary secretion, assessed by comparing the number of colonies grown ina culture medium “with” and “without” the addition of bile salts orhuman bile.

From these tests it emerged that a significant percentage of ingestedcells are able to overcome the gastric acidity barrier and pass beyondthe intestine.

This aspect is of particular importance as it guarantees that asufficient number of viable cells are able to reach the intestine, wherethe transformation of linoleic acid (LA) into its conjugated form (CLA)takes place.

Advantageously, the bacteria contained in the composition of the presentinvention are able to overcome the gastric barrier and duodenal transit,thus enabling colonisation of the gastrointestinal tract and theconversion of LA into CLA directly in situ, preferably in thegastrointestinal tract.

Although the mechanism of conversion from LA into CLA is not whollyclear at present, it seems that the bacterial strains adopt adetoxification strategy, transforming LA into its conjugated form CLAand removing LA from the environment in which it is present in excess.

In a preferred embodiment, the food or dietetic or pharmaceuticalcomposition of the present invention comprises, or alternativelyconsists of, at least one strain belonging to the species B. breve,selected from the group B. breve DSM 16604, B. breve DSM 16596 and B.breve DSM 20213. Advantageously, the food or dietetic or pharmaceuticalcomposition of the present invention comprises, or alternativelyconsists of, B. breve DSM 16604.

In another preferred embodiment, the food or dietetic or pharmaceuticalcomposition of the present invention comprises or consists of at leasttwo strains belonging to the species B. breve, selected from the groupB. breve DSM 16604, B. breve DSM 16596 and B. breve DSM 20213.

Advantageously, the food or dietetic or pharmaceutical composition ofthe present invention comprises, or alternatively consists of, B. breveDSM 16604 and B. breve DSM 16596.

Advantageously, the food or dietetic or pharmaceutical composition ofthe present invention comprises, or alternatively consists of, B. breveDSM 16604 and B. breve DSM 20213.

Advantageously, the food or dietetic or pharmaceutical composition ofthe present invention comprises, or alternatively consists of, B. breveDSM 16596 and B. breve DSM 20213.

In another preferred embodiment, the food or dietetic or pharmaceuticalcomposition of the present invention comprises, or alternativelyconsists of, at least three strains belonging to the species B. Breve,selected from the group B. breve DSM 16604, B. breve DSM 16596 and B.breve DSM 20213.

In another preferred embodiment, the food or dietetic or pharmaceuticalcomposition of the present invention comprises:

-   -   Bifidobacterium breve, deposited with the DSMZ and having the        accession number DSM 16604, filing date 20 Jul. 2004, depositor        Probiotical S.p.A., and/or    -   Bifidobacterium breve, deposited with the DSMZ and having the        accession number DSM 16596, filing date 21 Jul. 2004, depositor        Probiotical S.p.A., and/or    -   Bifidobacterium breve, deposited with the DSMZ and having the        accession number DSM 20213.

If two species of B. Breve are used, the ratio of viable cells iscomprised from 1:3 to 3:1, preferably 1:1.

If three species of B. Breve are used, the ratio of viable cells iscomprised from 3:1:1 to 1:1:1 (B. breve DSM 16604: B. breve DSM 16596:B. breve DSM 20213).

In a preferred embodiment, the food or dietetic or pharmaceuticalcomposition of the present invention comprises, or alternativelyconsists of, Bifidobacterium longum BL04, DSM 23233.

In another preferred embodiment, the food or dietetic or pharmaceuticalcomposition of the present invention comprises, or alternativelyconsists of, Bifidobacterium longum BL04, DSM 23233, in association withat least one of the following strains: Bifidobacterium breve DSM 16604,Bifidobacterium breve DSM 16596 or Bifidobacterium breve DSMZ 20213.

Advantageously, the food or dietetic or pharmaceutical composition ofthe present invention comprises, or alternatively consists of,Bifidobacterium longum BL04 DSM 23233 and Bifidobacterium breve DSM16604.

Advantageously, the food or dietetic or pharmaceutical composition ofthe present invention comprises, or alternatively consists of,Bifidobacterium longum BL04 DSM 23233 and Bifidobacterium breve DSM16596.

Advantageously, the food or dietetic or pharmaceutical composition ofthe present invention comprises, or alternatively consists of,Bifidobacterium longum BL04 DSM 23233 and Bifidobacterium breve DSM20213.

In another preferred embodiment, the food or dietetic or pharmaceuticalcomposition of the present invention comprises, or alternativelyconsists of, Bifidobacterium longum BL04, DSM 23233, in association withat least two of the following strains: Bifidobacterium breve DSM 16604,Bifidobacterium breve DSM 16596 or Bifidobacterium breve DSMZ 20213.

Advantageously, the food or dietetic or pharmaceutical composition ofthe present invention comprises, or alternatively consists of,Bifidobacterium longum BL04 DSM 23233, Bifidobacterium breve DSM 16604and Bifidobacterium breve DSM 16596.

Advantageously, the food or dietetic or pharmaceutical composition ofthe present invention comprises, or alternatively consists of,Bifidobacterium longum BL04 DSM 23233, Bifidobacterium breve DSM 16604and Bifidobacterium breve DSM 20213.

Advantageously, the food or dietetic or pharmaceutical composition ofthe present invention comprises, or alternatively consists of,Bifidobacterium longum BL04 DSM 23233, Bifidobacterium breve DSM 16596and Bifidobacterium breve DSM 20213.

In another preferred embodiment, the food or dietetic or pharmaceuticalcomposition of the present invention comprises, or alternativelyconsists of, Bifidobacterium longum BL04, DSM 23233, in association withat least three of the following strains: Bifidobacterium breve DSM16604, Bifidobacterium breve DSM 16596 and Bifidobacterium breve DSMZ20213.

Advantageously, the food or dietetic or pharmaceutical composition ofthe present invention comprises, or alternatively consists of,Bifidobacterium longum BL04 DSM 23233, Bifidobacterium breve DSM 16604,Bifidobacterium breve DSM 16596 and Bifidobacterium breve DSM 20213.

The ratio, in terms of viable cells, between Bifidobacterium Longum,BL04, DSM 23233 and the above-mentioned set of strains ofBifidocbacterium breve is comprised from 1:3 to 3:1, preferably 1:1.

In a preferred embodiment, the pharmaceutical composition containing thebacterial strains as specified above is indicated for use as amedication, preferably as an anti-inflammatory, in particular for thepreventive and/or curative treatment of intestinal disorders, diarrheaand inflammation of the colon, increasing the lean body mass, increasingthermogenesis, cancer prevention and protection against oxidativestress.

The composition of the present invention has application in thepreventive or curative treatment of conjugated linoleic aciddeficiences.

The composition of the present invention can be formulated in solid,lyophilised or dried form, for example in powder or granular form.

Moreover, one pharmaceutical form of interest is tablets or hard or softgelatin capsules.

Insofar as tablets are concerned, these may comprise an inner partcomprising the bacterial strains and an outer coating part. The coatingmay comprise water-soluble polymers and/or polymers able to withstandthe pH variations in the stomach and enable passage into the intestinaltract.

Another aspect of the invention relates to a method for the productionof conjugated linoleic acid.

The method involves a step wherein one or more bacterial strains, asspecified above, are cultivated/fermented in the presence of linoleicacid and the conjugated linoleic acid formed is subsequently isolated.

The method can be carried out in a laboratory or on an industrial scale.

Another aspect of the invention relates to the conversion from LA to CLAdirectly in the body once an individual has taken the composition of thepresent invention.

The conjugated linoleic acid that is obtained in larger quantity is theisomer [cis-9, trans-11 octadecadienoic acid], as compared to the isomer[trans-10, cis 12].

Another aspect of the invention relates to the use of at least onebacterial strain, as specified above, to prepare a composition for thetreatment of disorders or pathologies connected to a deficiency oflinoleic acid derivatives.

The Applicant conducted intense research activity on an extremely vastgroup of bacterial strains. All of the bacterial strains were tested andselected based on their ability/capability to convert LA into CLA.

The Applicant developed a culture medium suitable for carrying out theselection of strains and perfected the spectrophotometric techniquesused to carry out the selection of CLA-producing bacterial strains, withreadings at a wavelength of 233 nm.

Linoleic acid toxicity test and choice of culture medium. The strain B.breve DSMZ 20213 was selected as candidate for the linoleic acidtoxicity test. Said strain, in fact, is able to grow in the presence ofLA and displays good rates of conversion into CLA. Moreover, B. breveDSMZ 20213 was used for subsequent analyses as a positive control.Therefore, starting from the frozen form of said strain, it was possibleto revive it in three different culture media, which are specified intable 1.

After preparation, the media were sterilized in an autoclave at 121° C.for 15′. At the end of sterilization, the pH values of the media were6.60±0.10 at 25° C., 6.20±0.20 at 25° C. and 6.5±0.5 at 25° C.,respectively, for TPY, MRS and LAPTg broth. Revival was achieved byinoculating 1% of B. breve DSMZ 20213 into 10 ml of the three culturemedia with the addition of 1% cysteine chlorohydrate (5% sol.) andsubsequent incubation under anaerobic conditions using Gas-Packs at 37°C.±1° C. for 24 hours±1 hour. This operation was carried out through twosuccessive transplants to permit complete revival of the strain.

The strain thus revived was inoculated at 1% in 10 ml of the threedifferent culture media prepared as described above, with the additionof three solutions at different concentrations of linoleic acid,precisely 0, 0.5 mg/ml (Sigma-Aldrich cod.L1276), and then incubated inthe Gas-Pack at 37±° C. for 16 hours±1 hour.

In order to simplify the operation, three LA stock solutions wereproduced at 0 mg/ml, 50 mg/ml and 100 mg/ml, with the addition of 2%(v/v) Tween 80 to permit the formation of an emulsion and consequentincrease in the solubility of the linoleic acid dissolved using ananchor and magnetic stirrer.

Upon complete dissolution of the Tween 80, the solutions thus obtainedwere aliquoted and frozen at −25±1° C. following filtration with a 0.20μm filter.

A count was carried out by means of spectrophotometric reading at 600 nmat the start, at 6 hours and at the end of the incubation period. The pHwas recorded at the start and at the end of the incubation period.

Preparation of the Regression Line The regression line constructed forc9/t11 CLA concentrations of 320, 160, 80, 40, 20 and 10 μg/ml hexanedemonstrated that concentrations from 10 to 80 μg/ml were associatedwith a linear increase in the absorbance values at 233 nm (R²=1).

However, when the regression line was also calculated with theabsorbance value obtained for the concentration of 160 μg/ml, the linelost its linear pattern and became polynomial, thus precluding a precisequantification of the CLA concentrations for absorbances of between2.095 and 2.733. At concentrations equal to and greater than 320 μg/ml,the spectrophotometer gave non-quantifiable absorbance (>+3).

Therefore, the concentration of CLA present in the supernatants of theabsorbance cultures<2.095 can be directly calculated with the equationy=25.311x+0.0698. For samples that gave readings above 2.095, recoursewas made to the expedient of 1:2 serial dilutions.

Relying on a 1:2 serial dilution of the sample we thus brought theabsorbance values back within the interval which guarantees linearity,and hence the applicability of the equation. The data are shown in Table2.

To obviate the difficulty of assaying c9/t11 CLA (in the preparation ofthe first dilution necessary for calculating the regression line) due toits viscosity, and to guarantee maximum precision, we used a 2 μl fixedvolume pipette with glass capillaries.

The 2 μl sample of c9/t11 CLA thus obtained was then diluted with 1.450ml of hexane in a 2 ml test tube.

We thus obtained a starting concentration of 1280 μg/ml (densityc9/t11=0.903 mg/μl). 1:2 serial dilutions were made from thisconcentration until arriving at a concentration of 10 μg/ml. To limitevaporation of the hexane as much as possible, the various serialdilutions were performed in 200 μl micro test tubes previously filledwith 100 μl of hexane and then immediately closed. The various dilutionswere then obtained serially by adding 100 μl of the previous solution,starting from the mother solution at 1280 μg/ml.

At the end of the preparation of the various dilutions, we took aspectrophotometer reading of the optical density at a wavelength of 233nm, using quartz cuvettes with a nominal capacity of 100 μl. Saidoperation was carried out starting from the solution with the lowestconcentration (10 μg/ml).

The absorbance values thus obtained are shown in table 3.

Fatty Acid Extraction.

After a selection had been made as to the suitable culture medium and LAconcentrations to be used, they were thawed and reactivated.

The strain B. breve DSMZ 20213 was selected as a positive control. Thesame procedure was also applied on a sample of culture medium preparedwithout inoculum and with the addition of LA, to be used as a blanksample.

The TPY broth, after being supplemented with 1% of cysteinechlorohydrate (5% sol.), was selected as the culture medium for thesteps of reactivation and growth in the presence of 0.5 mg/ml of LA.

At the end of the incubation period, the culture broth and blank samplewere centrifuged at 5000 g for 5′ and the supernatants were aliquoted inamounts of 500 μl in 1.5 ml test tubes per microcentrifuge.

Then followed double hexane extraction according to the method of Yunget al., 2006 (Reference 1), with the modifications described below. Theextraction involved the addition of 500 μl of hexane to the aliquotedsupernatants and blank sample. The samples were then mixed by inversionfor 10′, with care being taken not to break the interface between thetwo steps, and then allowed to rest for 30′ at room temperature. At theend of the rest period, the samples were centrifuged at 2000 g for 5′ tofacilitate the operations of withdrawing the upper hexane phase. Oncethe upper hexane phase had been withdrawn and stored in a 2 ml testtube, the lower phase was again treated as described above with 500 μlof hexane. The upper phase was added to the previous one and mixed bytilting.

Spectrophotometric Analysis.

The CLA produced was quantified by means of a spectrophotometer readingof optical density using a wavelength of 233 nm, according to the methodof Barrett et al., 2007 (Reference 2) and Xu et al., 2008 (Reference 3).

Before the analysis was conducted, the spectrophotometer was zeroedagainst 100 μl of hexane using a quartz cuvette.

A reading was then taken for all of the samples, starting from theblank; care was taken to rinse the cuvette with hexane before proceedingwith the transfer of the sample.

For samples that gave absorbance values>+3, 1:2 serial dilutions inhexane were performed until reaching absorbance values such as to allowcalculation of the CLA concentrations by interpolation of the straightline Y=25.311 x+0.0698.

Spectrophotometric techniques for the quantification of CLA produced bybacterial cultures are known from Barrette et al., 2007. Thisspectrophotometric technique is based on a characteristic of conjugateddienes (in this case CLA) to possess a UV spectrum typical of this classof compounds, with an absorbance of 233 nm. Said method, in any case,permits a quantification of the CLA produced.

Linoleic Acid Toxicity Test and Selection of Culture Medium.

At the end of the incubation period (16 hours±1 hour, 37±1° C.), thetoxicity test conducted on the strain B. breve DSM 20213 using differentLA concentrations (0, 0.5 and 1 mg/ml) in three different culture media(TPY, MRS and LAPTg) made it possible to identify the most suitableculture medium for carrying out the subsequent screening step. TPY brothwas selected as a culture medium as it provided the environmentalconditions most favourable to the growth of Bifidobacteria in thepresence of LA. With TPY broth we found the least variability in countstaken both via spectrophotometer readings of optical density with awavelength of 600 nm and counts by the pour plate method, FIG. 1 andtable 4.

The differences observed in the use of TPY, MRS and LAPTg broth atdifferent LA concentrations (0, 035, 1 mg/ml) (FIG. 1) highlighted theimportance of the culture medium in studies regarding microbialmetabolism.

In fact, the use of TPY broth proved to be preferable to the use of MRSand LAPTg broth, as it gave the least variability in microbial growth atdifferent LA concentrations, as well as the highest counts, LAconcentrations being equal (0.5 and 1 mg/ml), compared to MRS and LAPTgbroth. The initial LA concentration selected was 0.5 mg/ml. Thisconcentration is in fact the preferable one for the LA-to-CLA conversiontests in MRS broth conducted in other studies (Barrett et al., 2007 andXu et al., 2008). The selection was dictated by the wish to compare, ina preliminary phase, the conversion rates obtained using TPY broth asthe culture medium and those reported in the literature with regard tothe use of MRS broth as the culture medium.

Selection of CLA-Producing Strains of Bifidobacteria.

At the end of the incubation period (16 hours±1 hour, 37±1° C.), theabove-specified bacterial strains and the negative control Lb. ReuteriDSM 20016 were submitted to spectrophotometric analysis at 233 nm, theprocedure necessary for quantifying the CLA produced. Table 5 shows theresults obtained with respect to the conversion of LA into CLA by thedifferent selected strains of Bifidobacteria.

The strain B. longum DSM 23233 showed to be the largest producer of CLA,with a production of 0.465 mg/ml, equal to an LA-to-CLA conversion rateof 92.93%. While the strain B. breve DSM 16604 showed to be a goodproducer of CLA, with a production of 0.393 mg/ml, equal to an LA-to-CLAconversion rate of 71.38%.

The CLA concentrations produced were calculated assuming that theculture media contained from the start variable quantities of LA and CLAdue to the presence of ingredients such as meat extracts, yeast extract,soybean components, etc. In this regard, we compared thespectrophotometer readings of optical density at the wavelength of 233nm for the different sterile media with and without LA at 0.5 mg/ml. Theresults obtained (table 6) revealed no significant difference betweenthe sterile medium with and without the addition of 0.5 mg/ml of LA.

The foregoing results confirmed the ability of the technique used toquantify exclusively the acidic component characterised by having twoconjugated double bonds, as in the case of CLA. Finally, the calculatedquantity of CLA present in the sterile culture media was subsequentlysubtracted in the calculation of the concentrations of CLA produced bymicrobial activity. Furthermore, the bacterial count performed by meansof spectrophotometer readings of optical density (wavelength 600 nm)showed a higher count for L. reuteri DSM 20016 versus the CLA-producingstrains, suggesting that toxicity related to the presence of LA isstrain specific.

Based on these results, it may be deduced that the most highly sensitivemicroorganisms are also the ones that are capable of achieving thegreatest conversion of LA to CLA, probably as a mechanism of defenceagainst the toxic element.

The tests performed as described above reveal a high percentage ofconversion for B. longum DSM 23233 (92.93%) and B. breve DSM 16604(71.38%) as compared to the positive control B. breve DSM 20213,relative to a study previously conducted using MRS broth (Barrett etal., 2007).

Therefore, the activity of conversion from LA to CLA performed byBifidobacteria could make this metabolite directly available in theintestinal lumen, where it could manifest its health-promotingproperties both locally and following its absorption.

The foregoing results reveal the ability of several strains ofBifidobacteria (in particular as regards the species B. longum and B.breve) to convert linoleic acid into conjugated linoleic acid.Accordingly, the use of such strains in the probiotic realm couldrepresent an efficacious and innovative means for controlling thedelicate balance in the dietary intake of omega-6/omega-3 EFAs.

The strains of Bifidobacteria capable of converting LA to CLA couldinteract in situ, along the gastrointestinal tract, with the essentialfatty acids taken in through the diet, potentially opposing thepro-inflammatory metabolism of the pathway of omega-6 fatty acids andproducing metabolites (CLA) with a possible nutraceutic role. In fact,even though the majority of the fats taken in through the diet aredigested in the small intestine, approximately 5-8 g of lipids reach thecolon every day. Furthermore, it has been estimated that humans expelaround 20 mg of LA through faeces. This suggests that LA is potentiallyusable as a substrate for the production of CLA. In addition, the recentisolation of strains of Bifidobacteria and Lactobacilli from the smallintestine of newborn babies extends the applicability of CLA-producingstrains of Bifidobacteria. These findings suggest a scope ofapplicability extending to nearly the entirety of the gastrointestinaltract, with the possibility of an action that is potentiallysimultaneous with lipid absorption, which occurs mainly in the upperregions of the small intestine. Finally, the applicability ofCLA-producing probiotic strains seems promising also because theanticarcinogenic effect of CLA could manifest itself at very lowconcentrations. In animal models, anticarcinogenic effects have beenobserved for quantities of CLA equivalent to 0.5-1% (w/w) of the dailydiet.

LIST OF REFERENCES

1) M. Y. Young et al., Technical Note: Improved Extraction Method withHexane for Gas Chromatograpic Analysis of Conjugated Linoleic Acids, J.Diary Sci. 89:90-94, American Diary Science Association, 2006.

2) E. Barrett et al., Rapid Screening Method for Analyzing theConjugated Linoleic Acid Production Capabilities of Bacterial Cultures,Applied and Environmental Microbiology, April 2007, p. 2333-2337,American Society for Microbiology.

3) H. Xu et al., Kinetics of microbial hydrogenation of free linoleicacid to conjucated linoleic acids, Journal of Applied Microbiology, ISSN1364-5072.

The invention claimed is:
 1. A method for treating a subject sufferingfrom a disorder or pathology connected to a deficiency in conjugatedlinoleic acid, the method comprising administering to said subject acomposition comprising an effective amount of a bacterial strainBifidobacterium longum deposited with the DSMZ on Jan. 12, 2010 underaccession number DSM 23233 (Bifidobacterium longum DSM 23233), therebytreating said subject for a disorder or pathology connected to adeficiency in conjugated linoleic acid.
 2. The method of claim 1,wherein the composition further comprises at least one bacterial strainselected from the group consisting of Bifidobacterium breve, depositedwith the DSMZ under accession number DMS 20213 (Bifidobacterium breveDMS 20213), Bifidobacterium breve, deposited with the DSMZ on Jul. 20,2004 under accession number DSM 16604 (Bifidobacterium breve DSM 16604)and Bifidobacterium breve, deposited with the DSMZ on Jul. 21, 2004under accession number DSM 16596 (Bifidobacterium breve DSM 16596). 3.The method of claim 2, wherein the composition comprises Bifidobacteriumbreve DSM
 16604. 4. The method of claim 2, wherein the compositioncomprises Bifidobacterium breve DSM
 16596. 5. The method of claim 2,wherein the composition comprises Bifidobacterium longum DSM 23233,Bifidobacterium breve DSM 16604 and Bifidobacterium breve DSM
 20213. 6.The method of claim 2, wherein the composition comprises Bifidobacteriumlongum DSM 23233, Bifidobacterium breve DSM 16596 and Bifidobacteriumbreve DSM
 20213. 7. The method of claim 2, wherein the compositioncomprises Bifidobacterium longum DSM 23233, Bifidobacterium breve DSM16604, Bifidobacterium breve DSM 16596 and Bifidobacterium breve DMS20213.
 8. The method of claim 2, wherein ratio of a number of viablecells of Bifidobacterium longum DSM 23233 to the number of viable cellsof all Bifidobacterium breve strains is between 1:3 and 3:1 in thecomposition.
 9. The method of claim 8, wherein the ratio is 1:1.
 10. Amethod for treating a subject suffering from inflammation, the methodcomprising administering to said subject a composition comprising aneffective amount of a bacterial strain Bifidobacterium longum DSM 23233,thereby treating said subject for inflammation.
 11. The method of claim10, wherein the composition further comprises at least one bacterialstrain selected from the group consisting of Bifidobacterium breve DMS20213, Bifidobacterium breve DSM 16604 and Bifidobacterium breve DSM16596.